PROJECT SUMMARY/ABSTRACT In children with congenital heart disease (CHD) there is an unidentified biological switch that drives a biologic transformation towards self-sustaining and progressive pulmonary arterial hypertension (PAH). In advanced disease, PAH secondary to CHD shares biologic similarities with other PAH groups. However, the signaling and metabolic derangements that drive early pulmonary vascular disease remain obscure, and currently available therapies largely fail to address the underlying pathologic origins of disease. My long-term research objective is to define the biochemical mechanisms by which aberrant pulmonary vascular hemodynamics initiate and drive pulmonary vascular dysfunction in CHD. The specific purpose of this application is to describe early metabolic and mechanotransductive signaling derangements in pulmonary vascular smooth muscle exposed to pulmonary overcirculation, and define their contributions to an abnormal vascular growth phenotype. Based on previously published work and novel preliminary data presented with this application, we hypothesize that exposure of pulmonary vascular smooth muscle to excessive pressure and blood flow results in sustained abnormalities of mechanotransductive signaling that perpetuate changes in cellular glutamine and cholesterol metabolism, promoting a dysregulated proliferative phenotype. In order to test this hypothesis, we are using a unique and clinically relevant animal model of CHD that recapitulates an early and progressive phase of disease that is poorly represented in other disease models. Our specific aims are to: 1) determine the role of altered glutamine and cholesterol metabolism in facilitating an abnormal proliferative phenotype in shunt pulmonary artery smooth muscle cells (PASMCs); 2) define the initiating and sustaining mechanisms that promote increased YAP signaling in shunt PASMCs and delineate the role of YAP in altered cellular metabolism and proliferation; and 3) evaluate metabolic biomarkers and therapeutic targets in a pre-clinical translational model of CHD. We will perform parallel C13 stable isotope resolved flux of glutamine and glucose, and conduct targeted manipulation of glutamine metabolism and cholesterol biosynthetic pathways to evaluate the impact on cellular proliferation. We will use a novel microfluidic cell culture bioreactor to assess the initiating and sustaining mechanical stimuli that induce the mechanosensitive transcriptional regulator YAP in shunt smooth muscle. We will also delineate the role of YAP in altered vascular smooth muscle metabolism and proliferation by targeted genetic knockdown of YAP and ChiP analysis. Finally, we will conduct translational evaluations of identified metabolic biomarkers and metabolic focused therapies in our model of CHD. The structured experience outlined in this proposal will solidify the knowledge and skills I require to transition to an independent research career and attain my long term scientific and career goals. Furthermore, the scientific understanding acquired will help us move towards more specific therapies for a clearly defined subset of pediatric patients with PAH, and will reveal important biologic features of a disease process that is largely studied in only its most advanced and severe forms.